DESCRIPTION: (provided by the applicant) Following the release of dopamine, its concentration in and around the synapse is rapidly reduced by the dopamine transporter (DAT), the major molecular target of cocaine and related psychostimulants. DAT requires extracellular sodium and chloride and couples the translocation of dopamine to the movement of these ions down their concentration gradients. Despite the cloning of DAT and related neurotransmitter transporters, its molecular structure and transport mechanisms are poorly understood. Progress in this area has been hampered by the lack of sufficient functional, purified protein and the inability to develop high-level expression systems for these proteins. Bacterial membrane proteins are generally more amenable to structural analysis and high-level expression than are their eukaryotic counterparts. We have recently identified an entire family of proteins in archaea and in bacteria that are homologous to DAT. Currently 39 proteins from 21 different organisms appear to fall into this family. None of these transporter proteins have been studied, and their substrates are unknown. In this CEBRA application, we propose to intensively explore the properties of a subset of these DAT archaeal and bacterial homologs in order to assess their suitability for use in further direct and indirect structural studies. Thus we propose the following specific aims: 1) To clone the 9 sodium- and chloride-dependent transporters from archaea and bacteria that are most similar to DAT. 2) To express these proteins heterologously in E. coli, and to assess and optimize levels of plasma membrane expression as weft as solubilization and purification conditions. 3) To express a candidate transporter(s), which based on studies in Aim 2 is suitable for further structural analysis, in Xenopus laevis oocytes to facilitate studies using electrophysiological techniques, focusing initially on sodium-dependent transient currents to determine whether the transporter(s) is inserted into the membrane and is functional. 4) To identify the substrates and/or non-substrate inhibitors of this transporter(s) by the measurement of substrate-induced currents. At the end of the period of support we will be in a position to choose a limited number of these archaeal and bacterial transporters for use in crystallization trials as a preliminary step towards obtaining a high-resolution structure. Moreover, we will also be poised to pursue biochemical and biophysical methods to acquire functional data and indirect structural information about these transporters. The resulting information will likely revolutionize our structural understanding of the function of DAT and related human neurotransmitter transporters, such as the serotonin and norepinephrine transporters that are targets for antidepressant drugs and cocaine, in a way that is only a distant prospect through continued work on the eukaryotic transporters alone.